Optimizing nonviral-mediated transfection of human intervertebral disc chondrocytes

Abstract

Background context

The use of viral vectors for transfection of human disc chondrocytes has been well documented. However, because of immunological and cell toxicity concerns, nonviral reagents may provide gene delivery to intervertebral disc (IVD) chondrocytes without these associated obstacles. Several studies have been done using nonviral delivery systems with varying degrees of success.

Purpose

The purpose of the study was to determine the efficiency, toxicity, and optimal conditions for gene delivery into human degenerative IVD cells via nonviral reagents in vitro.

Study design/setting

In vitro viral and nonviral gene transfer.

Patient sample

Human disc chondrocytes from 21 patients undergoing discectomy for trauma, disc herniation, and fusion for scoliosis or degenerative low back pain.

Outcome measures

Cell cytotoxicity and transfection efficiency as determined by microscopy, luciferase assay, and flow cytometry.

Methods

Seventeen lipid-based nonviral reagents coupled to DNA plasmids coding for luciferase were transfected into cultured chondrocytes. Cells were transfected with varying ratios of DNA plasmid to reagent, harvested at 48 hours and analyzed for transfection rates and cell viability. Transfections with adenoviral constructs were comparisons. The three most efficient reagents were then coupled to green fluorescent protein and the experiments repeated. The most efficient reagent after these experiments (LT1) was tested in standard chondrocyte-maintenance medium and a minimal medium mixture devoid of antibiotics, buffers, and amino acids. Finally, LT1 in minimal medium with various hyaluronidase treatments was tested. The most effective reagents and relative toxicity as measured by flow cytometry were analyzed using repeated measures analysis of variance.

Results

LT1 was most efficient and least toxic of nonviral reagents tested. LT1 had a mean percent survival of 78.1% versus 26.6% for TKO, 15.8% for T-Jurkat, and 70.8% in controls. Transfection was 1.5%. LT1 in minimal medium was significantly better than other reagents for both cell viability and transfection percentages. Minimal medium increased transfection with other reagents, yet cell viability with TKO and T-Jurkat was poor. Hyaluronidase had no effect on the viability of controls and decreased viability from 74.9% to an overall mean of 62.6% for all treatments. Transfection percentages increased from 1.8% without treatment to 15.2% with 40 units and 10.4% with four units of hyaluronidase given 24 hours before transfection and left in throughout the experiment. When treated at the time of transfection, efficiency was not significantly different to samples without hyaluronidase added. Additionally, hyaluronidase added 24 hours before transfection and washed out at the time of transfection significantly increased transfection percentages.

Conclusions

LT1 was the most efficient reagent in terms of transfection ability and cell toxicity compared with other reagents. Treatments in minimal medium yielded significant increases in transfection and no significant difference in toxicity as compared with controls. Hyaluronidase treatments improve transfection significantly but also increase toxicity. These results suggest that the nonviral reagent LT1 can be used to transfect IVD chondrocytes in vitro and may help facilitate gene transfection of IVD chondrocytes in vivo.

Introduction

Low back pain is the second most common complaint which brings a patient to a physician, and the most common cause of prolonged disability [1]. The cost related to the management of low back pain has been estimated to be between 90 and 100 billion dollars annually [2], [3]. Many cases are thought to be the result of degeneration of the intervertebral discs (IVDs) [4], [5], [6]. This process is associated with biochemical and biomechanical changes which can eventually result in morphologic findings and symptoms [7], [8], [9], [10], [11], [12], [13], [14]. Novel therapeutic regimens that can focus on this degenerative process are the subject of tremendous research interest [15], [16], [17]. The transfection of potentially therapeutic genes both in vitro and in vivo has been shown to increase proteoglycan production and increase water content of discs after degeneration [17], [18], [19], [20], [21], [22]. Several reagents have been reported, but the most commonly researched has been adenovirus [17], [19].

However, in vivo transfection of adenoviral vectors is limited by complications and potential toxicities such as direct cytotoxicity leading to apoptosis, immunologic response after transfection, and insertional mutagenesis and cell transformation. In a recent animal study, Wallach et al. documented paralysis after adenoviral-Transforming Growth Factor Beta (TGF β) intradural injection in high doses secondary to overexpression, immunological effect of the virus, or a combination of both [38]. Further, the use of viral based gene therapy on human subjects has been the source of recent controversy as a result of several isolated deaths [23], [24]. As a result, there is a clinical need for a nonviral reagent, if gene therapy is to be considered a viable option in the future.

Thus far, there is little data in the literature regarding the use of nonviral reagents in human disc chondrocytes. Early work using nonviral reagents for in vivo transfections was disappointing because of low transfection rates and short durations of gene expression in transfected cells [25], [26], [27]. However, Madry and Trippel [28] showed that articular cartilage chondrocytes could be successfully transfected in vitro using lipid-based reagents. Other transfection methods include the gene gun, transfer of naked DNA into living cells, and electroporation [17]. Although electroporation has shown recent promise, nonviral transfection of the intervertebral chondrocyte has remained elusive. Suitable reagents must be both nontoxic and efficient to deliver genes in sufficient quantity to be therapeutic.

The purpose of this study is to determine the efficiency, toxicity, and optimal conditions for gene delivery into human degenerative IVD chondrocytes via nonviral reagents in vitro. Nonviral reagents were also compared with adenovirus and a similar nonviral reagent already known to be effective in transfecting articular chondrocytes. The study was conducted in two phases. Phase 1 screened 17 lipid-based reagents to determine the most efficient reagents as determined by luciferase assay. Phase 2 involved several experiments to determine optimal conditions for in vitro transfection.